CN1159162A - Antithrombotic and non-hemorrhagic heparin-based compositions, method for their preparation and therapeutic applications - Google Patents

Abstract

Heparin compositions having an antithrombotic activity and virtually no hemorrhagic activity. The object of the invention is to eliminate the risk of bleeding associated with heparins while retaining their main properties. The compositions of the invention (S1, S2, S3) therefore consist of heparin moieties such as those obtainable by the in vitro neutralization of a heparin with a protamine. The invention also concerns a method for the preparation of these compositions which are useful in preparing medicaments.

Description

Contain antithrombotic formation and non-hemorrhagic heparin-based composition and method of making the same and treatment application

The present invention relates to contain the composition and method of making the same and the treatment application of heparin.

More particularly, the present invention relates to by the neutral heparin compostions that contains of protamine, said composition has anti-thrombosis activity but has obviously reduced hemorrhage and anticoagulating active.

People have known and have used the heparin many decades.Heparin is used to prepare the medicine with antithrombotic formation and/or anticoagulating active; This activity can be particularly useful as the preventative and therapeutic treatment of vein and artery thrombosis, perhaps as the coagulation activity in the prevention extracorporeal circulation.

People just know how to prepare low molecular weight heparin, this heparin has possessed anti-thrombosis activity before many decades, but its anticoagulating active has reduced.

However, for stepless heparin or low molecular weight heparin, hemorrhage risk remains the main concurrent problem based on the heparin therapy method.Greatly limited especially for the patient with hemorrhagic disposition thus, suffered from the patient of duodenal ulcer or gastric ulcer or carried out operating patient recently and use heparin, carrying out antithrombotic treatment by heparin in these patients may cause bleeding.

Therefore, when considering the considerable side effect relevant with this eternal hemorrhage risk, the superior function of heparin, promptly anti-thrombosis activity or anticoagulating active can not correctly be utilized.

When appearance was hemorrhage in utilizing the heparin therapy process, this treatment can be used the protamine sulfate that can produce heparin neutralization in the body.

Although it is not protamine so used for many years, fully aware of in the protamine with the mechanism of heparin.Recent relatively research shows simply: the comparable stepless heparin of low molecular weight heparin is neutralized to lower degree (" Prota minute e Neutralization of In Vitro Profile 0f HeparineDiffering in Source and Molecular Weight ", SE minute ARS INTHROMBOSIS AND IN HEMOSTASIS, vol.15, No.4,1989).

Therefore, one of problem to be solved by this invention is to reduce above-mentioned considerable hemorrhage risk, and this problem has limited the therapeutic use of heparin.

More properly, the objective of the invention is to eliminate as much as possible the hemorrhage risk relevant, meanwhile can keep its main performance, particularly its anti-thrombosis activity with heparin.

Thus, the purpose of this invention is to provide have very superior pharmacological properties particularly antithrombotic form the heparin compostions of performance, said composition is basic identical with the heparin compostions that uses at present, and does not show the major defect of considerable hemorrhage risk.

Another object of the present invention provides the method for this based composition of preparation, and this method is simple to operate and cheap, and can develop the treatment application of said composition.

The treatment that the invention still further relates to these compositionss is used.

But the heparin compostions of these purpose the application of the invention reaches, and described compositions has anti-thrombosis activity and do not have hemorrhagic activity basically.These compositionss are characterised in that basically to be made up of the heparin compositions that obtains with heparin in external by protamine.

The neutral heparin compositions of described protamine is to be understood that to by natural or fractionated heparin or by any composition of synthetic heparin derivative, the hemorrhage ability of this heparin by protamine or any analog with similar hemorrhage ability of reduction or its equivalent on and.

The present composition is primely by forming by the heparin compositions that utilizes stepless heparin of the external neutralization of protamine or low molecular weight heparin to obtain.

According to one embodiment of the invention, compositions is by wherein 25% forming greater than the heparin compositions of 20kDa for molecular weight less than 2.5kDa and 40% for molecular weight.

According to another embodiment of the present invention, compositions just is made up of less than the heparin compositions of 2.5kDa molecular weight.

In other embodiments, the molecular weight spectrum of heparin compositions depends on the neutral pattern of employed protamine.

The present composition is substantially free of protamine.

The present invention also provides the method for preparing above-mentioned composition, it is characterized in that comprising by protamine external in and the step of heparin.

The inventor is surprised to find: the hemorrhagic activity of heparin can be neutralized external, particularly can use protamine to neutralize, and meanwhile can keep its antithrombotic to form performance.

More properly, method of the present invention is included in the solution, and heparin is reacted with different heparin/protamine ratio with the protamine that particularly exists with the protamine salt form.

According to a preferred embodiment of the invention, heparin solution is preferably at room temperature mixed with the protamine saline solution, carry out the gained mixture centrifugal again and the collection supernatant.

According to the present invention, the term heparin solution is meant the solution of natural or classified heparin or synthetic heparin.

Protamine salt is made up of protamine sulfate primely.

According to the present invention, can use any have similar in and heparin ability and therefore reduce the protamine analog or the equivalent of hemorrhage ability.

Then can be with the supernatant lyophilizing.

Can use pending heparin of different proportion and the protamine that to eliminate the hemorrhage risk relevant substantially with heparin.

This method comprises to be utilized in protamine or the equivalent and the step of heparin, and preferred heparin/the protamine ratio is 2/1 to 1/2.

According to an embodiment of this method, heparin/protamine ratio is about 1/1.In the case, obtained containing wherein at least 25% molecular weight less than the molecular weight of 2.5kDa and at least 40% heparin compostions greater than the composition of 20kDa.

According to another embodiment of the present invention, heparin/protamine ratio is about 1/2.In the case, obtained containing basically the heparin compostions of molecular weight less than the composition of 2.5kDa.

The method according to this invention can not contained the heparin compostions of protamine.

The pharmacological research of heparin compostions of the present invention unexpectedly discloses: said composition does not have hemorrhagic activity basically, and keeps its antithrombotic to form performance.

Pharmacological research also unexpectedly discloses: the heparin compositions that the protamine neutralization obtains according to the present invention has anti-thrombosis activity, and this activity strengthens along with the increase of dosage, and can not increase its hemorrhage or anticoagulating active simultaneously.

Other experimental procedure can show: the present composition can more effectively suppress the hydrolysing activity of human leukocytes elastoser than stepless heparin.According to the present invention, suppress hemorrhage risk and can make that imagination is by non-intestinal path or by the path administration becomes possibility as aerocolloidal broncho-pulmonary in some bronchopneumopathy of treatment; It is too high that above-mentioned bronchopneumopathy may relate to leukocyte elastase, as acute respiratory distress syndrome, fibrocystic disease of pancreas and obstruction chronic bronchial pneumonopathy.

The present invention stablizes and nontoxic heparin compostions can be used for preparing the medicine that is used in the various treatments application.The application scenario that these are applied as heparin and standard derivant thereof comprises the situation according to the hemorrhage risk taboo heparin of patient's existence.Described compositions can be used for preparing the medicine of treatment and prevention vein or artery thrombosis or prevention extracorporeal circulation consolidation especially.

Therefore the invention still further relates to the pharmaceutical composition of the invention described above heparin compostions that comprises the treatment effective dose that makes up with pharmaceutically suitable carrier.

They can be as the anti-thrombosis drug compositions or be used to suppress the compositions of human leukocyte elastase hydrolysing activity.

The heparin compositions of these compositionss can be existed by the pharmaceutical acceptable salt that standard method obtains.

Pharmaceutical composition of the present invention is the ejection preparation by parenterai administration particularly primely.

For other application,, can provide the dosage form that is applicable to the broncho-pulmonary administration primely as suppressing leukocyte elastase.

Other features and advantages of the present invention will become obviously by reading the indefiniteness embodiment and the accompanying drawing that hereinafter provide, wherein:

Fig. 1 is the hemorrhagic activity comparison diagram of non-hemorrhagic heparin-based in stepless heparin, low molecular weight heparin and the heparin compostions of the present invention (S1, S2, S3);

Fig. 2 is the anti-thrombosis activity comparison diagram of stepless heparin, low molecular weight heparin and heparin compostions of the present invention (S1, S2, S3).

Embodiment

The product that uses: standard heparin (LEO), protamine sulfate (CHOAY) and low molecular weight heparin (" Enoxaparin ", commodity " Lovenox " by name are (PHARMUKA)).

-embodiment 1: preparation supernatant S1

Preparation 14.4ml titer is 72, and the standard heparin solution of 000IU (480mg) and 48ml titer are 48, the protamine sulfate solution of 000HAU.These solution at room temperature are mixed together.The ratio of heparin/protamine is 1: 1, that is to say, uses in the 1mg protamine sulfate and the lmg heparin.

With centrifugal 10 minutes of gained mixture, reclaim supernatant and lyophilizing.

-embodiment 2: preparation supernatant S2

Carry out according to the method for describing among the embodiment 1, use 9ml standard heparin solution (promptly 45,000IU, 300mg) with the 60ml protamine sulfate (promptly 60,000HAU).The ratio of heparin/protamine is 1/2, that is to say, uses in the 2mg protamine sulfate and the 1mg heparin.

-embodiment 3: preparation supernatant S3

Carry out according to the method for describing among the embodiment 1, use 4ml low molecular weight heparin solution (" Enoxaparin " (Lovenox)) (being 400mg) and 40ml protamine sulfate (promptly 40,000HAU).The ratio of heparin/protamine is 1/1, that is to say, uses in the 1mg protamine sulfate and the 1mg low molecular weight heparin.

Biological characteristic

-molecular weight distribution

Table I

The supernatant S1 that obtains by embodiment 1

-the molecular weight distribution represented by percent

Molecular weight	?????UV	????RI
			????＞20kDa	?????43.3	????47.3
????16-20kDa	?????2.7	????4.45
			????12-16kDa	?????4.5	????7.65
?????8-12kDa	?????9.7	????13.85
			?????5-8kDa	?????8.13	????11.8
????2.5-5kDa	?????6.36	????10.3
			????＜2.5kDa	?????25.16	????4.65
	????∑＝99.85	???∑＝100

The molecular weight distribution that the supernatant S2 that Table II is obtained by embodiment 2 is represented by percent

Molecular weight	????UV	????RI
			????＞20kDa	????0	????0
????16-20kDa	????0	????0
			????12-16kDa	????0	????0

????8-12kDa	????0	????0
			?????5-8kDa	????0	????0
????2.5-5kDa	????0	????0
			????＜2.5kDa	???100	???100
	??∑＝100	??∑＝100

The ultra-violet absorption spectrum of-supernatant S1 (embodiment 1)

Be diluted to 1/20 solution and have two absworption peaks at following wavelength place:

212nm:OD=3.47 and 271.5nm:OD=2.22

-titration supernatant S1 (embodiment 1)

Before the supernatant S1 that lyophilizing is made by embodiment 1, the 0.7ml supernatant soln of packing in each flask.In order to check reproducibility, carry out identical mensuration 12 times: the results are shown in down Table III:

Table III

The flask numbering	Heparin amount (mg/0.7ml) in each flask	??AZURE?A (IU/mg/ml)	????A-Xa (IU/mg/ml)	????A-IIa (IU/mg/ml)
					????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 ????9 ????10 ????11 ????12	????23.43 ????23.43 ????22.41 ????22.53 ????23.50 ????24.20 ????21.70 ????23.30 ????22.00 ????20.70 ????21.30 ????20.80	????84 ????83 ????84 ????83 ????86 ????83 ????86 ????83 ????85 ????83 ????85 ????80	????61 ????54 ????62 ????58 ????63 ????56 ????62 ????61 ????63 ????58 ????57 ????54	????32 ????29 ????32 ????30 ????30 ????28 ????29 ????31 ????30 ????32 ????28 ????30
????M±DS	????22±1.2	????84±2.2	????59±3.3	????30±1.4

People's such as AZUREA:Klein M.D. method A-Xa: (Hepadot Laboratoire Stago) A-IIa is measured in the timing of heparin: aminolysis method-protein determination

Measure respectively the supernatant S1 that makes by embodiment 1 and 2 and the protein among the S2 according to Pierce method (the breadboard reagent kit of Pierce).

The results are shown in down Table IV:

Table IV

Solution	Protein concentration (μ g/ml)
		????S1(2mg/ml)	????3.2
????S1(1mg/ml)	????＜1
		????S2(2mg/ml)	????29.9
????S2(1mg/ml)	????11.2
		????LOVENOX(2mg/ml)	????8.6
????LOVENOX(1mg/ml)	????6.7

The electrolyte composition that-electrolyte is formed among (mEq/l) supernatant S1 and the S2 is shown in down Table V:

Table V

	????Na	????K
			????S1	????24	????0.55
????S2	????18	????0.21

-determine pH

Table VI

Solution	????pH
		????S1	????5.57
????S2	????4.52

Pharmacological research

A. in the rat body to stagnate inductive venous thrombosis model and according to the research that experimentizes of hemorrhage guidance model

" Experimental venous thrombosis inrats treated with heparin and low molecular weight heparin fraction " according to people such as C.Doutremepuich, Haemostasis, 13, the method for describing among the 109-112 (1983) is studied.

A. treat model (induce thrombosis after two hours subcutaneous injection)

Carry out twice research according to following step:

T0: ligation caval vein

T0+2 hour: subcutaneous injection solution

T0+5 hour 30 minutes: induce hemorrhage

T0+6 hour: take out sample (blood and grumeleuse).

The result who carries out obtaining after the research for the first time is arranged in down among Table VII and the VIII:

Table VII

	Grumeleuse weight (mg)	??IHT(sec)	??CKT(sec)	????DTT(sec)
					Matched group	??5.54±1.54	??108±20	??19.6±1.3	????19.4±0.5
Heparin (2mg)	??1.76±0.53?? *	??420±00?? *	??180.0±0?? *	???180.0±0?? *
					????S1 ??(2mg)	??2.90±0.88?? *	??141±36	??23.2±1.8	????20.5±1.5
????S2 ??(2mg)	??4.19±1.07	??123±32	??21.4±2.1	????19.6±1.4
					?LovenoX ??(2mg)	??3.03±0.72?? *	??153±48	??25.2±2.1	????20.8±0.9
Heparin (1mg)	??4.18±1.06	??144±60	??25.9±2.6	????21.5±1.0
					????S1 ??(1mg)	??4.68±0.91	??122±28	??23.1±1.7	????20.5±1.5
????S2 ??(1mg)	??4.55±1.48	??123±34	??21.3±2.1	????19.7±1.2
					?Lovenox ??(1mg)	??4.84±0.94	??146±40	??21.6±2.0	????20.0±1.5

IHT: inductive bleeding time CKT: cephalin Kaolin time D TT: dilution thrombin time *=P＜0.05 (Mann Whitney test)

Table VIII

	Platelet (* 10 9/1)	Leukocyte (* 10 9/1)	Erythrocyte (* 10 12/1)
				Matched group	??538±220	??5.70±3.27	??7.40±0.39
Heparin (2mg)	??684±241	??4.25±1.80	??7.71±1.06
				???S1 ??(2mg)	??589±222	??4.07±2.06	??7.61±0.71
???S2 ??(2mg)	??546±155	??4.61±2.73	??7.53±0.97
				??Lovenox ??(2mg)	??606±113	??3.45±1.98	??7.81±0.99
Heparin (1mg)	??692±263	??5.02±3.25	??7.71±1.05
				???S1 ??(1mg)	??575±200	??4.23±1.68	??7.26±0.39
???S2 ??(1mg)	??600±242	??4.21±2.70	??7.46±1.15
				?Lovenox ??(1mg)	??621±188	??5.47±2.62	??7.88±0.81

Studies show that for the first time: in the heparin/protamine ratio that produces supernatant S1 is 1/1, when dosage is 2mg, with respect to stepless heparin and Lovenox (low molecular weight heparin), can access more considerable anti-thrombosis activity by the external neutral heparin of protamine, and anticoagulating active and hemorrhagic activity only there is very weak increase.

Supernatant S1 is to not effect of hemocyte.

Further, be that the supernatant S2 that 1/2 neutralization obtains does not have hemorrhagic activity by heparin/protamine ratio, but have the anti-thrombosis activity of reduction.

Research for the second time the results are shown in following table IX:

Table I X

Group		Grumeleuse weight (mg)	??IHT(s)	????CKT(s)	????DTT(s)
						The placebo group		??6.91±1.09	??126±49	????22±1.8	??19.6±1.0
??2mg	Heparin SN1 SN2 LOVENOX	??3.41±1.08 *??5.22±2.17 ??5.63±1.93 ??4.33±1.06 *	??＞420???? *??124±58 ??145±38 ??182±56?? *	??＞180?????? *??21.2±1.14 ??22.1±2.80 ??24.9±0.70	??＞180????? *??19.5±1.3 ??20.0±1.2 ??21.3±1.3
						??3mg	Heparin SN1 SN2 LOVENOX	??3.38±0.55 ??4.73±1.77 ??5.26±1.24 ??3.62±0.90 *	??＞420???? *??151±24 ??131±47 ??140±38?? *	??＞180?????? *??22.0±1.6 ??20.4±1.8 ??29.0±1.6	??＞180????? *??19.5±0.5 ??20.8±1.8 ??22.0±1.7
??4mg	Heparin SN1 SN2 LOVENOX	??2.75±0.91 ??4.09±1.10 ??4.72±2.33 ??3.33±0.98	??＞420???? *??155±43 ??136±48 ??198±76?? *	??＞180?????? *??22.5±1.9 ??18.6±5.7 ??50.9±3.9?? *	??＞180????? *??19.7±0.9 ??19.2±0.5 ??39.4±1.9? *
						??5mg	Heparin SN1 SN2 SN3 L0VENOX	??2.15±0.83 *??3.70±1.28 *??4.84±1.12 ??3.52±0.30 *??2.85±1.14 *	??＞420???? *??142±33 ??145±48 ??121±15 ??389±86?? *	??＞180?????? *??29.5±3.6?? *??22.0±1.3 ??20.8±1.8 ??＞180?????? *	??＞180????? *??24.2±9.3 ??20.3±0.7 ??19.6±1.75 ??＞180????? *
??10mg	Heparin SN1 SN2 SN3 LOVENOX	??0.98±0.82 *??3.15±1.21 *??4.09±1.16 *??2.29±0.40 *??1.44±0.48 *	??＞420???? *??171±64?? *??136±74 ??157±21 ??＞420???? *	??＞180?????? *??30.2±2.5?? *??23.8±1.7 ??27.0±2.0 ??＞180?????? *	??＞180????? *??32.6±6.9? *??20.0±0.7 ??23.0±1.0 ??＞180????? *

Clot wt.: the weight of experiment grumeleuse

IHT: induce the bleeding time

CKT: cephalin Kaolin time

DTT: dilution thrombin time

By the result who obtains as can be seen: the anti-thrombosis activity of composition S1 of the present invention, S2 and S3 increases along with the increase of dosage.

When we consider that dosage range is the dosage-effect curve of 2mg/kg to 10mg/kg, can be as seen from Figure 1, no matter the heparin type of the present invention's treatment how, the gained heparin compositions has and the similar hemorrhagic activity of matched group, even also be like this in very high dosage range.When comparing, do not carry out the neutral stepless heparin of protamine and low molecular weight heparin (Lovenox) has considerable hemorrhagic activity according to the present invention with heparin compositions of the present invention.

Fig. 2 shows that obtaining heparin compositions by the present invention has superior anti-thrombosis activity.Under the situation of composition S1 (heparin/protamine ratio is 1/1), this activity and the unneutralized heparin of the present invention active equally matched.

B. prophylaxis model (induce thrombosis before a hour through subcutaneous administration).

According to following step, utilize supernatant S1 (embodiment 1) to study:

T0: subcutaneous injection solution

T0+1 hour: stagnate and induce

T0+24 hour: take out sample (blood and grumeleuse).

Gained the results are shown in down Table X:

Table X

	Grumeleuse weight (mg)	??CKT(sec)	????DTT(sec)	????Ti(sec)
					Matched group	??5.13±1.03	??19.5±0.4	????18.7±0.6	??19.8±0.83
Heparin (4mg)	??3.40±0.70?? *	??20.7±0.4	????19.3±0.8	??19.8±1.30
					??S1 ??(4mg)	??3.23±0.61?? *	??20.5±1.1	????19.4±0.8	??19.3±0.83
??Lovenox ??(4mg)	??3.48±0.94?? *	??19.6±0.8	????19.7±0.9	??19.7±1.09

CKT: cephalin Kaolin time

DTT: dilution thrombin time

Ti:Titrarin (Stago Laboratory) time

*=p＜0.05 (mann Whitney test)

Gained is the result show: for the prevention purpose, after inducing thrombosis 24 hours, S1 has the anti-thrombosis activity suitable with Lovenox with heparin.

B. in the rat body with by producing the research that experimentizes of free yl induction thrombotic model

(list of references: Doutremepuich-in press-Annales de Cardiologieet Angiologie)

According to following step, utilize S1 (embodiment 1) to study:

(T0: subcutaneous injection solution)

T0+25 minute: injected dose was the rose-red of 5mg/kg

T0+30 minute: in first small artery, pass through the photochemically induced reaction free radical

T0+55 minute: inject the rose-red of same dose

T0+60 minute: in second small artery, induce free radical

T0+85 minute: inject the rose-red of same dose

T0+90 minute: in venule, induce free radical.

After the last thrombosis, intracardiac taking-up blood sample.

Stimulation time was located at 2 minutes, was located at observing time 10 minutes.

Obtain the result that provides among the following table XI:

Table X I

Small artery	?????????????????????????????T0＋30′
					Matched group	????S1(2mg/kg)	Heparin (2mg/kg)
Thromboembolism formation time (min)	????4.50±0.82	????9.68±0.44 *	????6.80±2.32
				Embolus quantity	????12.00±2.45	????4.00±3.56 *	????3.56±2.12 *
Small artery	??????????????????????????????T0＋60′
				Thromboembolism formation time (min)	????3.49±0.36	????9.81±0.25 *	????5.9±3.6
Embolus quantity	????7.33±0.47	????5.00±2.45	????4.56±3.6
Venule	?????????????????????????????T0＋90′
					Matched group	????s1(2mg/kg)	Heparin (2mg/kg)
Thromboembolism formation time (min)	????4.53±2.04	????3.68±2.06	?????????-
				Embolus quantity	????7.00±4.32	????4.00±1.41 *	?????????-

Thromboembolism formation time: by the time of isolating in the grumeleuse between first embolus and last embolus.

Embolus quantity: by the isolating embolus quantity of grumeleuse.

In this thrombotic model by free yl induction, compare with the placebo group, supernatant S1 (embodiment 1) has considerable anti-thrombosis activity, and this activity can continue 90 minutes (T0+90 minute).After 30 minutes and 60 minutes (T0+30 and T0+60 minute), this activity is higher than the heparin activity of injecting same dose.

C. in the rat body, induce the thrombotic model research that experimentizes according to the endothelial injury that causes by laser

(list of references: Vesvres, Haemostasis 1993,23,8-12)

A. study 1

Study according to following step: T0: substances T0+35 minute of subcutaneous injection 2mg/kg dosage: utilize laser beam to induce artery thrombosis.Be located at observing time 10 minutes.Gained the results are shown in following table XII:

Table X II

T0+35 ' (artery thrombosis)
					Matched group	??S1(2mg/kg)	Heparin (2mg/kg)
The laser irradiation number of times	??1.2±0.4	???2.0±1.4	?2.5±3.3
				The embolus number	??10.2±2.7	???1.5±0.7 *	?3.3±2.4 *
Thromboembolism formation time (min)	??6.3±1.8	???1.0±0.0 *	?2.1±1.8 *

S1 have with the injection same dose not in the anti-thrombosis activity suitable with heparin, and can reduce embolus quantity and shortening thromboembolism formation time with the statistics effective and efficient manner.

B. study 2

Study according to following step:

T0: the substances of subcutaneous injection 2mg/kg dosage

T0+1 hour: induce first artery thrombosis

T0+3 hour: induce second artery thrombosis

T0+6 hour: induce the 3rd artery thrombosis.

Be located at observing time 10 minutes.

Gained the results are shown in following table XIII:

Table X III

	????????????T0+1h		????????????T0＋3h		?????????????T0+6h
								????S1	????Hep	????S1	????Hep	????S1	????Hep
The laser irradiation number of times	??1.6±0.5	??1.6±0.5	??2.0±0.0	??1.6±0.5	??1.6±0.5	??1.0±0.0
							The embolus number	??3?0±1.0	??5.0±1.7	??5.3±3.5	??6.7±1.2	??8.3±3.0	??7.5±2.1
The thromboembolism formation time	??1.3±0.5	??2.6±1.2	??2.3±1.5	??3.0±1.0	??4.3±2.4	??3.0±1.4

S1 have with not by the suitable anti-thrombosis activity of the neutral heparin of protamine.

Briefly, above-described studies show that: all observed anti-thrombosis activity in the thrombotic model of three experiments, described three models serve as reasons and stagnate inductive vein model, by the artery thrombosis model of free yl induction with by the inductive artery thrombosis model of the endothelial injury that utilizes laser to cause.

According to the present invention, the heparin compositions that is obtained by low molecular weight heparin (" Enoxaparin " (Lovenox)) has the anti-thrombosis activity higher than the identical low molecular weight heparin without the protamine extracorporeal treatment, this activity is also than by the anti-thrombosis activity height without stepless heparin of protamine extracorporeal treatment, and no longer has hemorrhage risk.

Method of the present invention can be eliminated the hemorrhagic activity of heparin basically and keep its anti-thrombosis activity simultaneously with simple and inexpensive manner.

Claims (22)

1. have anti-thrombosis activity and do not have the heparin compostions of hemorrhagic activity basically, it is characterized in that said composition by in external by protamine and the heparin compositions that obtains of heparin form.

2. according to the compositions of claim 1, it is characterized in that said composition is made up of the heparin compositions that obtains by the stepless heparin of the external neutralization of protamine.

3. according to the compositions of claim 1, it is characterized in that said composition by in external by protamine and the heparin compositions that obtains of low molecular weight heparin form.

4. the compositions that one of requires according to aforesaid right is characterized in that said composition is made up of less than the heparin compositions of 2.5kDa at least 25% molecular weight wherein.

5. according to the compositions of claim 4, it is characterized in that said composition is made up of greater than the heparin compositions of 20kDa at least 40% molecular weight wherein.

6. according to the compositions of one of claim 1 to 4, it is characterized in that said composition is made up of less than the heparin compositions of 2.5kDa molecular weight.

7. according to the compositions of claim 5, it is characterized in that said composition is made up of the heparin compositions with the molecular weight distribution that provides as Table I.

8. according to the compositions of claim 7, it is characterized in that said composition is made up of the heparin compositions of the molecular weight distribution that provides as Table II.

9. any one compositions in requiring according to aforesaid right is characterized in that said composition is substantially free of protamine.

10. according to compositions any in the claim 1 to 9, it is characterized in that said composition has the performance that suppresses the human leukocyte elastase hydrolysing activity.

11. the preparation claim 1 to 10 in any method for compositions, it is characterized in that comprising with protamine external in and heparin.

12., it is characterized in that comprising mixing heparin solution and protamine saline solution, the mixture of centrifugal gained and the step of collecting supernatant according to the method for claim 11.

13., it is characterized in that described protamine salt is protamine sulfate according to the method for one of claim 11 and 12.

14., it is characterized in that heparin and protamine use with 1/1 ratio according to each method in the claim 11 to 13.

15., it is characterized in that heparin and protamine use with 1/2 ratio according to each method in the claim 11 to 13.

16., it is characterized in that stepless heparin is neutralized according to each method in the claim 11 to 15.

17., it is characterized in that low molecular weight heparin is neutralized according to each method in the claim 11 to 16.

18. each heparin compostions has anti-thrombosis activity and does not have purposes aspect the medicine of hemorrhagic activity basically in preparation in the claim 1 to 10.

19. pharmaceutical composition is characterized in that containing in the claim 1 to 10 with the effective dose of pharmaceutically suitable carrier combination each compositions.

20., it is characterized in that this pharmaceutical composition is the Injectable solution dosage form according to the pharmaceutical composition of claim 19.

21. be used to suppress the pharmaceutical composition of human leukocyte elastase hydrolysing activity, it is characterized in that containing with pharmaceutically suitable carrier combination as each compositions in the claim 1 to 10 of the effective dose of active component.

22., it is characterized in that this pharmaceutical composition is the dosage form that is applicable to the broncho-pulmonary administration according to the pharmaceutical composition of claim 21.